Intratumoral signaling via the cytosolic double-stranded RNA (dsRNA) receptors RIG-I and MDA-5 enhance antitumor immunity and can overcome resistance to cancer immunotherapy. Although BET proteins are established epigenetic drivers of immune escape mechanisms, their role in regulating RIG-I-like receptor (RLR) signaling remains undefined. Therefore, we investigated the role of BET proteins in modulating innate antiviral responses to cytosolic dsRNA in tumor cells and how BET inhibition combined with dsRNA stimulation influences tumor immunogenicity and recognition by T cells. To address this, human tumor cell lines were treated with the BET inhibitor JQ1 and transfected with the synthetic viral dsRNA analog poly(I:C) to stimulate innate antiviral signaling. We found that BET inhibition synergistically amplifies dsRNA-induced immunogenicity, as indicated by enhanced cytokine and chemokine production, JAK-STAT signaling, antigen presentation, and immunogenic cell death. Combined treatment of melanoma cells with JQ1 and poly(I:C) resulted in a marked, synergistic enhancement of recognition by autologous tumor-specific CD8+ TIL, immunogenic cell death and maturation of dendritic cells. Furthermore, an antiviral gene signature induced by poly(I:C) and enhanced by JQ1 correlated with immune infiltration and improved survival in larger patient cohorts across different types of cancer. Overall, our findings demonstrate that targeting BET proteins in tumor cells strongly potentiates dsRNA-induced antiviral responses and suggests that combining BET inhibitors with RLR agonists offers a pharmacological strategy to enhance antitumor T cells responses and improve the clinical efficacy of cancer immunotherapies.

Abstract Background Glucocorticoid prophylaxis is routinely used in oncology. Glucocorticoids potent immunosuppressive and anti-inflammatory properties raise concerns regarding potential impairment of antitumor immune responses. Preclinical and retrospective studies suggest glucocorticoids may impair antitumor immunity, though these findings are subject to significant bias and confounding factors. Understanding the immunomodulatory effects of prophylactic glucocorticoids, isolated from other therapies, is critical to bridging the knowledge gap on their influence on antitumor immunity. Methods We performed a post hoc, retrospective analysis using cryopreserved PBMCs and plasma from patients with treatment-naïve, early stage HER2+ breast cancer, prior to neoadjuvant therapy. Of 192 patients, 129 had no prior glucocorticoid exposure and 63 received 24 mg prophylactic betamethasone before sampling. High-dimensional flow cytometry assessed monocytes, dendritic cells, and T-cell subsets. Plasma proteins were evaluated using the Olink Target 96 Immuno-Oncology proximity extension assay. Results Glucocorticoid exposure was consistently associated with a distinct immune profile. Exposed patients exhibited increased CD163+ monocytes and reduced intermediate, non-classical, and HLA-DRhi classical monocytes, plasmacytoid DCs, and conventional CD1c+ DCs. T-cell alterations included enrichment of CXCR4+ subsets, particularly terminally differentiated CD8+ T-cells, with reductions in naïve, central memory, CD27+ effector memory, and CXCR3+ populations. Inflammatory plasma proteins (IFN-γ, CCL19, CCL2, IL-12, IL-6, Granzyme A/B, CXCL10, CXCL9) were diminished, whereas IL-10 and CXCL13 were elevated. Glucocorticoid-associated immune alterations were heterogeneous across patients, highlighting interindividual variability in observed post-exposure immune states. Conclusions Prophylactic glucocorticoid exposure reshapes the circulating immune landscape, altering monocytes, dendritic cells, naïve and memory T-cell subsets, as well as key inflammatory proteins.

BET inhibitors (BETi) have shown potential to augment tumor immunogenicity in melanoma. However, conflicting evidence exists regarding their precise mechanism of action, and their overall impact on melanoma immunogenicity and antitumoral T cell responses remains unclear. To address this, human melanoma cell lines treated with JQ1 and/or IFNγ were investigated for gene and protein expression changes in key pathways governing immunogenicity and cocultured with autologous tumor-infiltrating lymphocytes (TIL) with known antigen-specificity. JQ1-induced proteome-wide alterations were examined using mass spectrometry-based cellular thermal shift assay (MS-CETSA), which revealed that JQ1 broadly impacts melanoma immunogenicity by regulating IFN signaling, antigen processing and presentation, and innate immune signaling pathways. More specifically, JQ1 enhanced JAK1/STAT1 signaling and upregulated components of the HLA class I (HLA-I) antigen processing and presentation machinery (APM), increased MART-1 expression while concomitantly dampening tumoral expression of PD-L1, IDO1, and HLA class II (HLA-II). Functionally, JQ1 markedly improved tumor recognition by autologous MART-1- and neoantigen-specific CD8+ TIL, while dampening CD4+ TIL activation through the downregulation of Cathepsin S (CTSS). Preliminary results using JQ1-treated melanoma cells in a mixed lymphocyte-tumor cell culture (MLTC) markedly enhanced TIL proliferation and resulted in a T cell product enriched for CD8+ T cells. These findings reveal how the pleiotropic effects of BETi on melanoma cells broadly boost their immunogenicity towards CD8+ T cells and uncover novel pathways that might be therapeutically exploited to enhance CD8+ T cell-mediated anti-tumor immunity in ex vivo and in vivo approaches to cancer immunotherapy.

“Pro-senescence therapy”, which triggers both permanent cell cycle arrest and an immune response, is a controversial new strategy for cancer treatment. To assess this strategy in melanoma, we performed a high throughput microscopy-based senescence screen utilizing a panel of melanoma cell lines with different driver mutations and a collection of clinical and experimental drugs. We found that vemurafenib and trametinib, which inhibit BRAFV600E and MEK1/2, respectively, induced senescence in some but not all BRAF-mutant cell lines. In contrast, palbociclib, BKM-120 and crizotinib, which inhibit CDK4/6, PI3K, and MET/ALK/ROS1, respectively, triggered senescence in most cell lines, irrespective of BRAF/NRAS mutation status, and overcame intrinsic and acquired vemurafenib resistance. The combination of palbociclib and crizotinib synergized to further enhance the senescence response in all cell lines irrespective of BRAF/NRAS mutation status, increased the expression of SASP factors, such as IL-1α and β, and HLA class I and other markers for recognition by NK and T cells. Further, this combination caused a significant increase in CD8+ T cells and pro-inflammatory macrophages in the tumor microenvironment and a marked reduction of mouse melanoma tumor growth that was dependent on CD8+ T cells, suggesting increased immune surveillance. Our findings suggest that pro-senescence therapy based on concomitant inhibition of both CDK4/6 and MET/ALK/ROS1 could be developed further as an alternative treatment strategy for melanoma. Significance Pro-senescence therapy based on combined targeting of CDK4/6 with Palbociclib and MET/ALK/ROS1 with Crizotinib inhibits melanoma tumor growth through anti-tumor immune response activation, providing an alternative treatment strategy for malignant melanoma. ### Competing Interest Statement LGL is cofounder of MyCural Therapeutics AB and has ownership interests in this company. KGW is cofounder and shareholder of Aprea Therapeutics, a company that develops novel anticancer therapy including APR-246 (Eprenetapopt). KGW has previously obtained research funding and salary from Aprea Therapeutics. KGW is board member of MyCural Therapeutics AB. LBo is founder of T-Hope Nordics AB and has ownership interests in this company. Knut and Alice Wallenberg Foundation, KAW 2013.0093 Cancerfonden, 24 3901 Pj, 21 1817 Pj, 19 0561 Pj

Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell–to–myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell–derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m -knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell–mediated mechanism that drives the development of MDSCs during tumor immune escape.

Oxidative stress, that is, an unbalanced increase in reactive oxygen species (ROS), contributes to tumor-induced immune suppression and limits the efficacy of immunotherapy. Cancer cells have inherently increased ROS production, intracellularly through metabolic perturbations and extracellularly through activation of NADPH oxidases, which promotes cancer progression. Further increased ROS production or impaired antioxidant systems, induced, for example, by chemotherapy or radiotherapy, can preferentially kill cancer cells over healthy cells. Inflammatory cell-derived ROS mediate immunosuppressive effects of myeloid-derived suppressor cells and activated granulocytes, hampering antitumor effector cells such as T cells and natural killer (NK) cells. Cancer therapies modulating ROS levels in tumors may thus have entirely different consequences when targeting cancer cells versus immune cells. Here we discuss the possibility of developing more efficient cancer therapies based on reduction-oxidation modulation, as either monotherapies or in combination with immunotherapy. Short-term, systemic administration of antioxidants or drugs blocking ROS production can boost the immune system and act in synergy with immunotherapy. However, prolonged use of antioxidants can instead enhance tumor progression. Alternatives to systemic antioxidant administration are under development where gene-modified or activated T cells and NK cells are shielded ex vivo against the harmful effects of ROS before the infusion to patients with cancer.

PDF file - 75K, Supplementary figure 1 depicts the absolute lymphocyte counts in patients during ipilimumab treatment. Supplementary figure 2 depicts the correlation analysis of different dellular populations

Supplementary Figure 2 from DNAX Accessory Molecule-1 Mediated Recognition of Freshly Isolated Ovarian Carcinoma by Resting Natural Killer Cells

<p>Supplementary Table S1 - PDF file 80K, Supplemental table containing additional authentication data</p>

<p>Legend for Supplemental Figures S1-S4</p>

<p>Melanoma cells induce MDSC-like cells through COX-2/PGE2 production.</p>

PGE2 receptor EP3 is responsible for the activation of STAT-3 signaling on MDSC-like cells.

<p>PDF file - 207K, Table S1. Cell line authentication. Table S2. Antibodies used for flow cytometry.</p>

NK cell cytotoxicity against 4T1mCr-1 cell line.

Supplementary Figure Legends 1-5 from Immature Immunosuppressive CD14+HLA-DR−/low Cells in Melanoma Patients Are Stat3hi and Overexpress CD80, CD83, and DC-Sign

Supplemental table containing additional authentication data.

PDF file - 213K, This figure shows the gating strategy used for analyzing the arginase staining

Tumor-educated MDSC-like cells are potent suppressors of autologous T cells.

<p>Supplementary Table S1 - PDF file 80K, Supplemental table containing additional authentication data</p>